Accurate chromosome segregation is essential for the propagation of species and the viability of cells, and is driven by a complex microtubule-based structure called the spindle. Intensive biochemical, genetic, and proteomic efforts provide an extensive catalogue of proteins that participate in spindle organization and spindle-dependent chromosome movement. However, these efforts don't reveal the molecular mechanisms that ensure faithful chromosome segregation in mammalian cells. Recently, we showed that the most common cause of chromosome mis-segregation in human tumor cells is the persistence of kinetochore-microtubule (K- MT) attachment errors. However, our understanding for how K-MT attachments are regulated to promote error correction remains starkly incomplete. We don't understand how different molecular components create a coherent output to fine-tune K-MT attachment stability during cell cycle transitions. We also don't understand how aneuploidy influences genome stability and cell survival. It is our goal in the forthcoming funding period to combine biochemical methods and live cell imaging to test models and determine the mechanisms of the regulation of K-MT attachments in mitosis. Understanding these mechanisms could lead to new therapeutic approaches for cancer treatment. The specific aims of this proposal are, 1.) To use live cell imaging to determine the biochemical changes underlying the transition from prometaphase to metaphase; 2.) To combine live cell imaging with molecular tools to test the model that changes in the copy number of single genes is sufficient to undermine faithful chromosome segregation by disrupting K-MT attachment dynamics; 3.) To quantify the genetic damage caused by persistent K-MT attachment errors; and 4.) To test mechanisms that suppress aneuploid cell growth in live animals.